The membrane-associated phospholipid biosynthetic enzymes are important because their activities are involved in structural as well as functional properties of cell membranes. In the yeast Saccharomyces cerevisiae the enzyme activities in the reaction sequence phosphatidic acid - greater than CDP-diacylglycerol - greater than phosphatidylserine - greater than phosphatidylethanolamine - greater than phosphatidylmonomethylethanolamine - greater than phosphatidyldimethylethanolamine - greater phosphatidylcholine are coordinately regulated to the synthesis of phosphatidylinositol. This application will focus on the regulation of four of the enzyme activities in this pathway, namely, CDP-diacylglycerol synthase, phosphatidylserine synthase, phosphatidylinositol synthase, and the phospholipid N-methyltransferases. CDP-diacylglycerol synthase and the phospholipid N-methyltransferases will be purified to homogeneity and will be characterized with respect to enzymological, chemical, and physical properties. Purified preparations of phosphatidylserine synthase and phosphatidylinositol synthase are already available for the project. The purified enzymes will be reconstituted into well defined unilamellar phospholipid vesicles to study activity modulation by phospholipids, water-soluble metabolites, and by phosphorylation/dephosphorylation. Antibodies specific for each of the enzymes will be prepared and used as probes to study the regulation of enzyme formation in wild type and mutant strains of S. cerevisiae grown under various conditions. Enzyme regulation will also be studied in clones containing the structural genes encoding for these enzymes. The results found with S. cerevisiae should be relevant to higher eucaryotic organisms.